Circadian clocks control a wide variety of fundamental cellular, physiological, and behavioral processes in eukaryotic organisms. The molecular machinery that permits the measurement of time is referred to as the circadian clock and its output as circadian rhythms. Our long-term goal is to understand the molecular and biochemical mechanisms of circadian clocks. The filamentous fungus Neurospora crassa, which has one of the best understood circadian clock systems, offers a powerful experimentally-accessible system for exploring the clock mechanism at molecular levels. Like circadian oscillators in the higher eukaryotic organisms, the Neurospora oscillator consists of an autoregulatory negative feedback loop. In this core negative feedback loop, two WHITE COLLAR proteins (WC-1 and WC-2) are the positive elements that form a WC complex that activate the transcription of the frequency (frq) gene. The negative elements are FRQ and FRH, a FRQ- interacting RNA helicase, which form a complex that inhibits the WCC activity. The transcriptional and posttranscriptional regulation of FRQ plays central roles in the control of the clock. In this proposal, we aim to address several fundamental questions of the clock mechanism. In Specific Aim 1, we will determine how phosphorylation of FRQ regulates its activity and its structural conformation. This study will help establish a biochemical mechanism critical for the circadian negative feedback process in Neurospora. Rhythmic activation of clock gene transcription is an essential process in eukaryotic circadian clocks. We recently discovered CATP as a critical clock component that controls frq transcription by affecting chromatin structure at the frq locus. In Specific Aim 2, we will determine the mechanism for how CATP regulates frq transcription by regulating the chromatin structure. Antisense RNAs are present at the frq locus in Neurospora and per loci in animals. We recently demonstrated that the expression of the frq antisense RNA qrf is essential for clock function. In Specific Aim 3, we will determine the mechanism for how qrf transcription regulates frq expression by transcriptional interference. Together, these objectives take advantage of a well-established model system to address three fundamental questions that are critical for our understanding of eukaryotic circadian clocks and will elucidate the genetic, biochemical, and molecular mechanism of the Neurospora clock. Because of the conservation between the Neurospora and animal clocks, our results will provide important insights into eukaryotic clock mechanisms.